Orotidine 5'-monophosphate decarboxylase (ODCase) is a key enzyme in the de novo pyrimidine biosynthetic pathway. This pathway is essential for the biosynthesis of building blocks for both DNA and RNA, and thus inhibitors of ODCase could have significant therapeutic value. Despite recent significant advances, it is not understood what factors are important in binding to and catalysis by ODCase. The lack of such knowledge is a critical deficit because understanding these factors is essential to the design of potent inhibitors for this enzyme. Our long-range goal is to understand the catalytic mechanism of ODCase and to design potent inhibitors of ODCase based on the mechanism. The objective of this application, which is a step in pursuit of that goal, is the identification of structural components of the substrate and its analogs that are important for binding and catalysis. The central hypothesis of the application is that the active site is structured to preferentially bind nucleotides with large matched dipole moment (as a result of an overall negative charge around the heterocyclic ring). The rationale for the proposed research is that, once the structural features determining tight binding between the enzyme and nucleotides are identified, novel potent inhibitors can be designed, which could serve as lead compounds in the design of drug candidates. The hypothesis will be tested by pursuing the following two specific aims: 1) Determine how the negative charge on the nucleotide base and the relative positions of the pyrimidine moiety to the phosphoribose moiety affect the binding of substrate analogues; and 2) Determine structural requirement for substrate binding and to eventually identify amino acid residues interacting with the substrate carboxylate group. PUBLIC HEALTH RELEVANCE: Orotidine 5'-monophosphate decarboxylase (ODCase) is a key enzyme in the de novo pyrimidine biosynthetic pathway. This pathway is essential for the biosynthesis of building blocks for both DNA and RNA. Inhibition of the biosynthesis of these building blocks has been exploited in the development of antiviral, antitumor, and antimalarial drugs.